Two new deaza analogues of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 1), 7-deaza-EHNA (6) and 1,3-dideaza-EHNA (11), were synthesized and evaluated for adenosine deaminase (ADA) inhibitory activity ...and compared with EHNA, 1-deaza-EHNA (2), and 3-deaza-EHNA (3). Substitution of a methine group for a nitrogen atom in the 7-position of the purine moiety of EHNA produces a dramatic drop in the inhibitory activity (Ki = 4 X 10(-4) M) whereas compounds 2 and 3 are still good inhibitors (Ki = 1.2 X 10(-7) M and 6.3 X 10(-9) M respectively). EHNA and its deaza analogues so far synthesized were also tested in vitro for their antiviral and antitumor activity in a range of cellular systems. EHNA and 1-deaza-EHNA are equiactive as inhibitors of human respiratory syncytial virus (HRSV) replication (MIC = 6.25 micrograms/mL) while the other compounds are inactive. On the other hand, all the examined compounds displayed an antitumor activity comparable to that of the reference compound 1-beta-D-arabinofuranosyladenine (ara-A), 7-deaza-EHNA being the most active of all. The results obtained showed that there is no correlation between adenosine deaminase inhibition and antiviral or antitumor activity in this series of compounds. 3-Deaza-EHNA, the most active inhibitor of ADA among the EHNA deaza analogues, greatly potentiates the antitumor activity of ara-A in vitro. In vivo activity was observed only when the two compounds were used in combination.
The tritiated analogue of 2-chloro-N6-cyclopentyladenosine (CCPA), an adenosine derivative with subnanomolar affinity and a 10,000-fold selectivity for A1 adenosine receptors, has been examined as a ...new agonist radioligand. 3HCCPA was prepared with a specific radioactivity of 1.58 TBq/mmol (43 Ci/mmol) and bound in a reversible manner to A1 receptors from rat brain membranes with a high affinity KD-value of 0.2 nmol/l. In the presence of GTP a KD-value of 13 nmol/l was determined for the low affinity state for agonist binding. Competition of several adenosine receptor agonists and antagonists for 3HCCPA binding to rat brain membranes confirmed binding to an A1 receptor. Solubilized A1 receptors bound 3HCCPA with similar affinity for the high affinity state. At solubilized receptors a reduced association rate was observed in the presence of MgCl2, as has been shown for the agonist 3HN6-phenylisopropyladenosine (3HPIA). 3HCCPA was also used for detection of A1 receptors in rat cardio myocyte membranes, a tissue with a very low receptor density. A KD-value of 0.4 nmol/l and a Bmax-value of 16 fmol/mg protein was determined in these membranes. In human platelet membranes no specific binding of 3HCCPA was measured at concentrations up to 400 nmol/l, indicating that A2 receptors did not bind 3HCCPA. Based on the subnanomolar affinity and the high selectivity for A1 receptors 3HCCPA proved to be a useful agonist radioligand for characterization of A1 adenosine receptors also in tissues with very low receptor density.
2-Chloro-N6-cyclopentyladenosine (CCPA) was synthesized as a potential high affinity ligand for A1 adenosine receptors. Binding of 3HPIA to A1 receptors of rat brain membranes was inhibited by CCPA ...with a Ki-value of 0.4 nM, compared to a Ki-value of 0.8 nM for the parent compound N6-cyclopentyladenosine (CPA). Binding of 3HNECA to A2 receptors of rat striatal membranes was inhibited with a Ki-value of 3900 nM, demonstrating an almost 10,000-fold A1-selectivity of CCPA. CCPA inhibited the activity of rat fat cell membrane adenylate cyclase, a model for the A1 receptor, with an IC50-value of 33 nM, and it stimulated the adenylate cyclase activity of human platelet membranes with an EC50-value of 3500 nM. The more than 100-fold A1-selectivity compares favourably with a 38-fold selectivity of CPA. Thus, CCPA is an agonist at A1 adenosine receptors with a 4-fold higher selectivity and 2-fold higher affinity than CPA, and a considerably higher selectivity than the standard A1 receptor agonist R-N6-phenylisopropyladenosine (R-PIA). CCPA represents the agonist with the highest selectivity for A1 receptors reported so far.
![2-Chloro-N6-[3H]cyclopentyl...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0028-1298/small "2-Chloro-N6-[3H]cyclopentyladenosine ([3H]CCPA)--a high affinity agonist radioligand for A1 adenosine receptors")